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Home > Vascular Annual Meeting > ofstein_greater_saphenous

Greater Saphenous Vein Is a Novel Source of Endothelial Colony Forming Cells

Richard Ofstein, Janak Bhavsar, Reza Saadatzadeh, Michael C. Dalsing, Shaoib Shafique, Michael P. Murphy.
Indiana University, Indianapolis, Ind.

OBJECTIVES: Endothelial colony forming cells (ECFCs) have been isolated and characterized from human umbilical cord blood. ECFCs are highly proliferative, differentiate into endothelial cells, and in vivo participate in vasculogenesis, all of which are hall marks of a true endothelial stem cell. Given these characteristics, ECFCs hold great promise for cell based therapies for tissue ischemia, however their potential may be limited by immunological responses to allogeneic cells. Using the same novel cloning cell culturing techniques, we have been able to isolate ECFC’s from the endothelium of adult greater saphenous vein (GSV).

METHODS: GSV was collected from vein stripping procedures and endothelium was removed using a cell scraper. Viable cells were enumerated and plated on six-well plates pre-coated with collagen type I with 1x103 cells per well. At day 3-8 of culture, cells were selected from colonies using a sterile cloning cylinder, replated and expanded to confluency on collagen type I. Cultured cells were detached and incubated with antibodies to cell surface markers: CD105, CD31, CD133, CD45, CD144, CD34, and KDR for immunophenotyping. Using the clonogenic assay, one CD144±CD45- cell was placed per well of a 96 well plate coated with type 1 rat tail collagen. On day 14 the wells containing greater than 50 cells were quantified. Tube formation was assessed by plating 6x103 cells in Matrigel and incubating for 24 hours.

RESULTS: GSV endothelial cells gave rise to ECFC colonies at day 3-8 of culture [Figure 1].

Using fluorescent activated cell sorting, cultured cells expressed CD144, CD31, KDR, CD105, and did not express CD45, CD34, or CD133 [Figure 2].

 

The clonogenic assay demonstrated that 26% of cultured cells divided, and of these, 30% gave rise to greater than 10³ cells, comparable to cord blood ECFCs [Figure 3].

When plated in matrigel, GSV ECFCs formed microtubules [Figure 4].

CONCLUSIONS: These results demonstrate that adult greater saphenous vein contains a population of highly proliferative cells that express endothelial specific antigens and are capable of forming microtubules in vitro. GSV-ECFCs therefore represent an ideal population of autologous cells for future therapy of cardiovascular disease.

AUTHOR DISCLOSURES: R. Ofstein, None; J. Bhavsar, None; R. Saadatzadeh, None; M.C. Dalsing, None; S. Shafique, None; M.P. Murphy, None.